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Resolution of the type material of the Asian elephant, Elephas maximus Linnaeus, 1758 (Proboscidea, Elephantidae)


Abstract and Figures

The understanding of Earth's biodiversity depends critically on the accurate identification and nomenclature of species. Many species were described centuries ago, and in a surprising number of cases their nomenclature or type material remain unclear or inconsistent. A prime example is provided by Elephas maximus, one of the most iconic and well-known mammalian species, described and named by Linnaeus (1758) and today designating the Asian elephant. We used morphological, ancient DNA (aDNA), and high-throughput ancient proteomic analyses to demonstrate that a widely discussed syntype specimen of E. maximus, a complete foetus preserved in ethanol, is actually an African elephant, genus Loxodonta. We further discovered that an additional E. maximus syntype, mentioned in a description by John Ray (1693) cited by Linnaeus, has been preserved as an almost complete skeleton at the Natural History Museum of the University of Florence. Having confirmed its identity as an Asian elephant through both morphological and ancient DNA analyses, we designate this specimen as the lectotype of E. maximus. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the data set identifier PXD000423. © 2013 The Linnean Society of London
Content may be subject to copyright.
Resolution of the type material of the Asian
elephant, Elephas maximus Linnaeus, 1758
(Proboscidea, Elephantidae)
1Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster
Voldgade 5-7, 1350 Copenhagen, Denmark
2Natural History Museum, Cromwell Road, London SW7 5BD, UK
3Department of Bioinformatics and Genetics, Swedish Museum of Natural History, SE-10405
Stockholm, Sweden
4Department of Zoology, Stockholm University, SE-10691 Stockholm, Sweden
5Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois
61801, USA
6Jesus College, Turl Street, Oxford OX1 3DW, UK
7Lincoln School of Humanities, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK
8The Roslin Institute, University of Edinburgh, Midlothian EH25 9RG, UK
9Department of Zoology, Swedish Museum of Natural History, SE-10405 Stockholm, Sweden
10Natural History Museum of Florence, via Romana 17, 50125 Florence, Italy
11Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of
Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark
12Ancient DNA Laboratory, Murdoch University, South St, Perth, Western Australia 6150, Australia
Received 24 June 2013; revised 20 August 2013; accepted for publication 20 August 2013
The understanding of Earth’s biodiversity depends critically on the accurate identification and nomenclature of
species. Many species were described centuries ago, and in a surprising number of cases their nomenclature or type
material remain unclear or inconsistent. A prime example is provided by Elephas maximus, one of the most iconic
and well-known mammalian species, described and named by Linnaeus (1758) and today designating the Asian
elephant. We used morphological, ancient DNA (aDNA), and high-throughput ancient proteomic analyses to
demonstrate that a widely discussed syntype specimen of E. maximus, a complete foetus preserved in ethanol, is
actually an African elephant, genus Loxodonta. We further discovered that an additional E. maximus syntype,
mentioned in a description by John Ray (1693) cited by Linnaeus, has been preserved as an almost complete skeleton
at the Natural History Museum of the University of Florence. Having confirmed its identity as an Asian elephant
through both morphological and ancient DNA analyses, we designate this specimen as the lectotype of E. maximus.
*Corresponding author. E-mail:
Zoological Journal of the Linnean Society, 2014, 170, 222–232. With 3 figures
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232222
The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the data
set identifier PXD000423.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232.
doi: 10.1111/zoj.12084
ADDITIONAL KEYWORDS: Albertus Seba – ancient DNA – ancient proteins – Carl Linnaeus – John Ray
– lectotypification – Loxodonta.
The International Code of Zoological Nomenclature
(ICZN; 1999) establishes the starting date for zoologi-
cal nomenclature as 1 January 1758, the year when
Edition 10 of the Systema Naturae of Carl Linnaeus
(1758) was published. References cited by Linnaeus
form an integral part of the description of his species,
and material that the descriptions and/or illustrations
of the cited authors were based on is considered
syntypic (forming part of the name-bearing type
series), whether or not it was examined by Linnaeus
and whether or not it still exists (ICZN Article 72.4.1).
Thus, both Linnaeus’ own specimens and descriptions,
as well as those of the earlier authors he cited, are
of equal status for zoological nomenclature. Like
his predecessors, Linnaeus (1758) did not distinguish
between Asian and African elephants. Amongst the
authors he cited in his description of Elephas
maximus, the figure in Gesner (1551), reproduced in
Aldrovandi (1616), very likely represents an African
elephant, whereas figures in Jonston (1650), pls 7–9
show an Asian elephant (Supporting Information S1),
thus suggesting that E. maximus might have a com-
posite type series. The African elephant was estab-
lished as two separate species later: Elephas africanus
Blumenbach, 1797 (currently Loxodonta africana) and
Elephas cyclotis Matschie, 1900 (currently Loxodonta
A most remarkable specimen referred to by
Linnaeus (1764) in his description of the Swedish
King Adolf Fredrik’s collection, is the well-preserved,
near-complete, body of an elephant foetus in a spirit
jar, held today at the Swedish Museum of Natural
History (NRM) in Stockholm. This specimen was ini-
tially owned by the Dutch West India Company,
which operated in West Africa, the Americas, and
the Pacific. The foetus later became part of Albertus
Seba’s natural history collection, one of the richest of
its time (Supporting Information S2). Between 1734
and 1765, Seba (1734, 1759, 1765) published descrip-
tions with plates of his specimens in his ‘Thesaurus’.
The elephant foetus is clearly illustrated in vol. 1
(plate 111, fig. 1; 1734, here reproduced in Fig. 1A),
and it is recorded (p. 175) as originating in Africa.
The Swedish sovereign and his wife bought the
specimen either at the April 1752 auction of Seba’s
collection, or shortly thereafter from one of the other
purchasers (Lovén, 1887; Engel, 1961; Boeseman,
1970). The elephant foetus was kept at Drottningholm
Palace from 1773 to 1801 and was finally placed in
what became the NRM (Fig. 1B). The provenance of
the sample is without doubt: NRM inventories record
the specimen as coming from Drottningholm, origi-
nally from Seba’s cabinet, and it was mentioned by
Linnaeus (1764) in his published account of the King’s
Despite its importance as a syntype of E. maximus,
the species identity of the foetus has been questioned.
Sundevall (1857) and Lönnberg (1904) both believed
that, although it was labelled Elephas maximus, the
specimen was identifiable as an African elephant,
genus Loxodonta. Together with the African origin
for the specimen, noted by Seba (1734), this again
would suggest that E. maximus has a composite type
Here, we sought to further resolve the identity of
the foetal syntype specimen of E. maximus. We used
ancient protein and DNA sequencing (see Methods) to
conclusively establish the taxonomic identity of Seba’s
elephant foetus as an African rather than an Asian
elephant. Second, in examining John Ray’s (1693)
text cited by Linnaeus, reporting the description of an
elephant skeleton, we found archival evidence indi-
cating that this specimen still exists today in the
collection of the Natural History Museum of Florence.
Using morphological and molecular evidence, we
confirm that it is the skeleton of an Asian elephant
eligible for designation as the lectotype of E. maximus
The elephant foetus, kept in the collection of the
Swedish Museum of Natural History of Stockholm
(catalogue number NRM 532062), was gently removed
from its jar and laid on a tray. The specimen presented
an extended thoracic−abdominal longitudinal opening.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
Operating through the opening, a small, approxi-
mately 5×5×3mm, oesophagus fragment and the
terminal portion of a cartilaginous rib of similar size
were removed and transferred to Copenhagen for
ancient protein analysis. A second rib fragment was
collected and stored using the same procedure for
aDNA analysis in Stockholm.
For ancient protein analysis, oesophagus and rib
subsamples, of 64 and 39 mg (wet weight) respectively,
were cut in a chemical hood, on a sterile Petri dish,
using a single-use, disposable, sterile scalpel. A nega-
tive control containing no sample was prepared and
analysed following the same procedure used for the
ancient samples. Samples were dried in a centrifu-
gal evaporator to remove residual ethanol. Tryptic
peptides were generated using a filter-aided sample
preparation protocol (Wisniewski et al., 2009). Protein
digestion was started by adding 4 μL 0.5 μg/μL
sequencing-grade trypsin solution (Promega) and incu-
bating at 37 °C overnight at pH 7.50−8.00. Tryptic
peptides were then acidified with 10% trifluoroacetic
acid to a final concentration of 0.2−0.8% to reach a
pH <2.00 and immobilized by C-18 solid phase extrac-
tion on Stage Tips (Rappsilber, Ishihama & Mann,
2003) as previously described (Cappellini et al., 2012).
The liquid chromatography−mass spectrometry (LC-
MS) system consisted of an EASY-nLC system
(Thermo Scientific, Odense, Denmark) connected to a
Q Exactive mass spectrometer (Thermo Scientific,
Bremen, Germany) through a nano electrospray ion
(ESI) source. Five μL of each peptide sample were
auto-sampled onto and directly separated in a 15 cm
analytical column (75 μm inner diameter) in-house
packed with 3 μm C18 beads (Reprosil-AQ Pur, Dr.
Maisch), using a 130-min linear gradient from 5 to 26%
acetonitrile followed by a steeper linear 20-min gradi-
ent from 26 to 48% acetonitrile. Throughout the
gradients a fixed concentration of 0.5% acetic acid
and a flow rate of 250 nL min−1 were set. The effluent
from the high-performance LC was directly electro-
sprayed into the mass spectrometer by applying
2.0 kV through a platinum-based liquid-junction. The
settings used were as described for the ‘sensitive’
acquisition by Kelstrup et al. (2012). The mass spec-
trometry proteomics data have been deposited in the
Figure 1. The elephant foetus, NRM 532062 from the Swedish Museum of Natural History, referred to by Linnaeus.
A, reproduction of the foetus illustration in Figure 1, Plate 111, vol. 1 of Albertus Seba’s ‘Locupletissimi rerum naturalium
thesauri accurata descriptio,et iconibus artificiosissimis expressio, per universam physices historiam . . . (1734). B, recent
photograph, and C, radiograph of the same specimen. Scale bar in panel B equals approximately 10 cm.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
ProteomeXchange Consortium (http://proteomecentral via the PRIDE partner reposi-
tory (Vizcaíno et al., 2013) with the data set identifier
Raw files of the two processed samples, generated
during spectra acquisition, were merged and searched
on a workstation [64523 tandem mass spectrometry
(MS/MS) spectra, 31561 and 32962 from oesophagus
and the rib samples, respectively, were submitted for
analysis] using the MaxQuant algorithm v.
(Cox & Mann, 2008) and the Andromeda peptide
search engine (Cox et al., 2011), initially against the
target/reverse protein list included in the L. africana
reference proteome (downloaded from UniProtKB,, on 29 February 2012), and then, in
a separate search, against the complete list of pro-
teins available on the same date in UniProtKB after
taxonomic restriction to E. maximus. In every search,
spectra were also matched against the common con-
taminants such as wool keratins and porcine trypsin,
downloaded from UniProt.
To identify more genus-diagnostic tryptic peptides,
we adopted a proteogenomics approach (Sanders
et al., 2011) that: (1) extracted, from the L. africana
genome (loxAfr3.67 downloaded from Ensembl
Genome Browser,, the DNA
sequences coding for the tryptic peptides confidently
identified by matching the spectra from the Seba
elephant against the African elephant reference
proteome; (2) extended for 120 bases each of the ends
of the identified coding regions; (3) assembled, if pos-
sible, the DNA sequences previously generated into
short contigs, to obtain the coding DNA sequence for
the ancient proteins identified on the basis of several
contiguous peptides; (4) mapped a set of shotgun
Illumina DNA reads, generated at the Roslin Institute
from two modern Asian elephants, against the
L. africana DNA extended reads and short contigs
coding the peptides identified in the Seba foetus;
and (5) filtered to retain alternate, homozygous,
nonsynonymous single nucleotide polymorphisms
(SNPs) in the positions coding for the ancient foetus
tryptic peptides, allowing us to identify which of the
peptides identified in the ancient sample had genus-
diagnostic potential. Detailed description of this pro-
cedure is reported as Supporting Information S3.
African savannah elephant sequences of HBB/D,
IARS,SPR, GSTP and COL6A3 (full names are listed
in Table 1) were identified in the National Center
for Biotechnology Information (NCBI) Loxodonta
africana genome Trace Archives (http://www.ncbi using MegaBlast (Zhang
et al., 2000) and also NCBI GenBank entries
(COL6A3: XM_003417935, GSTP: XM_003419394,
IARS: XM_003421084, SPR: XM_003413564,
HBB/D: XM_003421554). African savannah elephant
sequences were aligned with their Asian elephant
(E. maximus) homologues, obtained as described in
Supporting Information S3.5–6, and primers were
designed at conserved regions to amplify genus-
diagnostic nonsynonymous codon mutations, using
the software PRIMER3 (
primer3/input.htm; Rozen & Skaletsky, 2000). One or
two sets of primers were designed to amplify each
region (Supporting Information Table S3.4).
PCR and sequencing reactions were performed
using M13 tailed primers (M13 forward tail sequence
at the 5end of the forward primers and M13 reverse
tail sequence at the 5end of the reverse primers),
following the protocol by Ishida et al. (2011). Six
African savannah elephants, five African forest
elephants, and five Asian elephants were sequenced
for each of the five genes (Supporting Information
Table S3.5). Sequences were aligned to L. africana
predicted mRNA (GenBank numbers as above) and
the coding regions were translated using the ExPASy-
translate tool (; Artimo
et al., 2012) to identify and confirm genus-diagnostic
amino acid differences.
Sequences of 4258 bp of mtDNA (from MT-ND5 to
control region) were aligned for 653 Loxodonta
(GenBank accession numbers: JQ438119–JQ438771;
Ishida et al., 2013) and 73 E. maximus (unpubl. data).
Three short regions that contained genus-diagnostic
nucleotide sites were identified, and primers were
designed with conserved sequences at 3ends, using
the software PRIMER3 (Supporting Information
Table S3.6; Rozen & Skaletsky, 2000).
In anticipation of their application to ancient DNA,
primers were tested using highly diluted modern
elephant DNA. Human genomic DNA was also tested
as a negative control to establish that primers would
not amplify human DNA, which sometimes may con-
taminate ancient samples. All work involving modern
DNA was performed at the University of Illinois at
Urbana-Champaign, with primers ordered separately
for use by the ancient DNA laboratory (Ishida et al.,
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
Given the degraded nature of DNA in ancient speci-
mens, pre-PCR laboratory work was performed in a
dedicated ancient DNA laboratory at the Swedish
Museum of Natural History (Stockholm). Negative
controls and repeated (once or more) amplifica-
tion were used to minimize the effects of potential
contamination and to monitor damaged sites. DNA
extraction was performed on a small, approximately
5×5×3mm, piece of soft-tissue from the elephant
foetus, using a QIAamp DNA micro kit (Qiagen) and
following the manufacturer’s protocol for isolation of
genomic DNA from tissues. To minimize alteration of
the ancient specimen, a bone microcore, 5 mm diam-
eter, was collected from the left humerus of the Flor-
ence elephant skeleton using a dentist’s trephine
(Asa Dental, Italy). Approximately 100–200 mg of
bone powder obtained from the microcore were used
for DNA extraction, following a modified version of
protocol C in Yang et al. (1998). The extraction buffer
included 1 M urea instead of sodium dodecyl sulphate
and the post-lysis extraction buffer was concentrated
using a 30 K MWCO Vivaspin filter (Sartorius). Silica
spin columns were used for final DNA purification.
For the Seba elephant foetus, three pairs of
mitochondrial DNA primers mentioned above (Sup-
porting Information Table S3.6) were used to amplify
DNA from the specimen. Additionally, two short frag-
ments of the nuclear genes Biglycan (BGN) and
Phosphorylase kinase alpha subunit 2 (PHKA2) that
contain diagnostic sites that distinguish African forest
from African savannah elephants were also targeted,
using primer pairs BGN-s1F2/BGN-s1R2 and PHK-
s1F/PHK-s1R, respectively (Supporting Information
Table S3.7; Ishida et al., 2011). For PCR amplification
of these DNA fragments, the M13 tailed primers were
used to increase amplicon length and the quality of the
sequences (Supporting Information Table S3.7; Ishida
et al., 2011). We also included M13 tails in some of the
mitochondrial DNA primer pairs.
For the Florence skeleton, 741 bp of mitochondrial
DNA were amplified in seven overlapping fragments,
using primer pairs from Barnes et al. (2007). This
region includes the 3end of the cytochrome bgene
(MT-CYTB), two tRNA genes: mitochondrially encoded
tRNA threonine (MT-TT) and mitochondrially encoded
tRNA proline (MT-TP), and the first hypervariable part
of the control region (CR1).
PCR reactions had a final concentration of 1 × PCR
buffer, 0.2 μM of each primer, 200 μM deoxyribonu-
cleotide triphosphates, 2.5 mM MgCl2, 0.1 mg mL−1
bovine serum albumin, 2 units HotStarTaq DNA poly-
merase (Qiagen), purified water, and 2 μL DNA extract
Table 1. Genus-diagnostic tryptic peptides supporting the attribution of the ancient elephant foetus to Loxodonta. Genus-distinctive amino acids are in bold.
Their position refers to the protein sequence identified by the accession number reported.
number Protein name
(Da) Charges PEP
aa substitution
Haemoglobin sub. Beta/Delta 19 2132.033 2,3,4 7.35E-15 161.24 7 52 (D)
2 EAALVDVVNDGVEDLR G3TPD1 Glutathione S-transferase P-like 16 1712.858 2 7.92E-12 141.46 6 93 (A)
3LYLINSPVVR G3TAH7 Isoleucyl-tRNA synthetase,
10 1172.692 2 0.003211 102.61 1 630 (M)
4NMMFQVLAAEEPTVR G5E6U0 Sepiapterin reductase-like 15 1734.843 2 3.34E-05 119.01 2 185 (V)
Collagen alpha-3(VI) 16 1643.013 2,3 3.44E-06 123.38 9 208 (M)
PEP, posterior error probability; aa, amino acid; *the corresponding Elephas-distinctive amino acid substitutions are reported in parentheses.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
in a total volume of 25 μL mix. PCR conditions
included an initial denaturation step of 10 min at
95 °C, followed by 55 cycles of denaturation of 30 s at
94 °C, annealing of 30 s at 50 or 52 °C (depending on
the primer pair; Supporting Information Tables S3.6,
S3.7), extension of 30 s at 72 °C, and a final extension
step of 7 min at 72 °C. Amplicons were purified with
Exonuclease I and FastAP Thermosensitive Alkaline
Phosphatase. Sequencing was performed in both
directions on an ABI3130xl (Applied Biosystems)
automated sequencer at the Molecular Systematics
Laboratory, at the Swedish Museum of Natural
History (Stockholm). Sequences were assembled in
GENEIOUS 5.0.1 (
Morphological comparison of foetal organisms can be
challenging. Nevertheless, preliminary inspection sug-
gested that the foetus could be assigned to Loxodonta,
based on comparatively large ears and two finger-like
processes on the tip of the trunk (Fig. 1B; Shoshani,
2000). X-ray images of the specimen (Fig. 1C) were also
taken to enable counting of the ribs and the thoracic,
lumbar, and caudal vertebrae, which differ in number
between African and Asian elephants (Shoshani,
2000). However, not all of these elements could be
individually distinguished on the X-ray images owing
to the incomplete development of the animal.
To resolve the identity of the Seba elephant foetus, we
analysed DNA and sequenced proteins (Cappellini
et al., 2012) using a shotgun proteomics approach from
a small section of the oesophagus and the terminal
portion of a cartilaginous rib. Nano-liquid chromatog-
raphy coupled with high-resolution MS/MS was used
to generate a spectra data set from tryptic peptides
and identify, as genus-diagnostic markers, the non-
identical homologous ones present in publicly avail-
able protein lists for both elephant genera. The total
mass of proteins recovered before digestion was 126
and 90 μg, whereas after digestion it was 72 and 60 μg,
for oesophagus and rib extracts, respectively. However,
as the nuclear genome of E. maximus was not publicly
available at the time of analysis, and the list of publicly
available protein sequences from this species is
quite limited, at only c. 200, only seven proteins and
48 peptides identified in the Seba elephant sample
were available for both L. africana and E. maximus.
Despite such a limited set of homologous proteins and
peptides, we did nevertheless identify one variable
tryptic peptide from haemoglobin subunit Beta/Delta
(Opazo et al., 2009): FFEHFGDLSTAE52AVLHNAK,
with start-end amino acid positions 41–59 in both
UniProt accessions P02085 and P02084, for L.
africana and E. maximus, respectively. This protein
had a putative genus-diagnostic amino acidic substi-
tution at position 52, namely Glu (E) in the African
and Asp (D) in the Asian elephant. As the spectrum
reported in Figure 2 demonstrates, the assigned
peptide sequence clearly indicated that the Seba foetus
had the African elephant version of the peptide.
Parameters supporting the identification of the genus-
diagnostic peptide and haemoglobin subunit Beta/
Delta are reported in Table 1 and Supporting
Information Table S3.8, respectively.
The proteogenomics approach returned four nonsy-
nonymous SNPs falling within regions coding for the
peptides identified in the Seba elephant sample. At
each of the four additional genus-diagnostic peptides,
the Seba elephant matched Loxodonta and not
Elephas. Proteomics parameters supporting the iden-
tification of the additional four genus-diagnostic
peptides, and of the corresponding L. africana proteins
that they are part of, are reported in Tables 1 and S3.8,
respectively, whereas examples of their MS/MS spectra
are visible in Supporting Information Figures S3.1–4.
PCR-amplification and Sanger-sequencing of the
DNA regions coding for the five genus-diagnostic
tryptic peptides identified in the elephant foetus,
applied to 16 modern elephant individuals of diverse
geographical provenance, confirmed the Loxodonta/
Elephas diagnostic capacity of these peptides (Sup-
porting Information Table S3.5).
Three regions of mtDNA were successfully amplified
and sequenced for the Seba foetal elephant specimen,
using primer pairs Ele-ND5-F1/R1, Ele-ND5-F3/R3,
and Ele-cytB-1/R1. The three sequences were com-
pared to Loxodonta and Elephas sequences. All diag-
nostic sites matched Loxodonta and not Elephas
sequences. Two of the Seba elephant mtDNA frag-
ment sequences (Ele-ND5-F1/R1 and Ele-ND5-F3/R3)
included a site diagnostic for the west-central
subclade, and matched the character state present
in the west-central subclade at all informative sites
(Supporting Information Table S3.9; Ishida et al.,
2013). By contrast, three to six character state mis-
matches separated the Seba foetus sequence from the
seven other major mtDNA subclades identified in
African elephants (Ishida et al., 2013). The geographi-
cal distribution of this subclade notably corresponds
to the west-central region of Africa in which the
Dutch West India Company had conducted its African
operations since the first half of the 17th century.
Identification of the species of the elephant foetus as
African savannah or African forest elephant was not
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
Figure 2. Tandem mass spectrum, generated from the analysis of the Seba elephant foetus sample, supporting identification of the Loxodonta version of the
genus-diagnostic tryptic peptide F41FEHFGDLSTAE52AVLHNAK59, from haemoglobin subunit Beta/Delta, (UniProt accession number: P02085). Δmz
12 11
87 129 043 the presence of Glu in position 52. Peaks in the spectrum indicate the detected mass-to-charge ratios of peptide fragmentation
products. Peaks that support assignment of the spectrum to the amino acidic sequence in the figure are colored (blue and red indicate b- and y-series fragments,
respectively). Resulting fragmentation pattern is overlaid on the sequence.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
possible because nuclear loci could not be sequenced
(Ishida et al., 2011). Despite the short length of tar-
get amplicons and attempts to optimize the PCR
reactions/conditions, the degraded DNA of the foetus
failed to amplify for two nuclear loci with sites diag-
nostic between forest and savannah elephants.
Having established that the Seba foetus was
Loxodonta, we exhaustively scrutinized the refer-
ences cited by Linnaeus (1758). In one of these refer-
ences, John Ray’s ‘Synopsis Methodica Animalium
Quadrupedum et Serpentini Generis’ (1693: 131), we
found a detailed description of the skeleton of an
elephant that Ray had observed while visiting
Florence between 14 July and 1 September 1664
(Supporting Information S4). His description (Sup-
porting Information S5), and that of his travelling
companion Phillip Skippon (1732), do not contain
information sufficient to identify the specimen as
either Asian or African elephant. However, anatomical
details, together with other historical sources, clearly
indicate that the skeleton is the one still on display
at the Natural History Museum of the University
of Florence (Fig. 3A). Records indicate that these
remains belong to an elephant that died in Florence on
9 November 1655 (Supporting Information S6), as
reported in a drawing by the artist Stefano Della Bella
(Fig. 3B). This was probably the itinerant performing
elephant known as ‘Hansken’, born in Ceylon (today
Sri Lanka), and portrayed in 1637 by the Dutch artist
Rembrandt van Rijn (Supporting Information S7;
Slatkes, 1980). After dissection, the skeleton was
mounted and exhibited in a room at the Uffizi Gallery
(Targioni Tozzetti, 1780) until, in September 1771 the
Grand Duke of Tuscany ordered the relocation of
all specimens of scientific interest into the newly
constituted Museum of Physics and Natural History,
now Natural History Museum. The continuous pres-
ence of the elephant remains at the Uffizi Gallery
first, then at the Natural History Museum, is docu-
mented by several bibliographical sources (Ray, 1673,
1693; Skippon, 1732) and museum inventories (Sup-
porting Information S6). In addition, the current
wooden replica of the sternum (Fig. 3C) conforms
to Ray’s indication that this element was missing,
whereas further anatomical peculiarities mentioned
by Skippon (1732) can be observed today in the
Florence skeleton: fusion of the second and third
Figure 3. A, elephant skeleton MZUF-734 at the Natural History Museum of the University of Florence. B, drawing by
Stefano Della Bella: ‘Elephant died in Florence on November 9th 1655’, inset (translation from Italian by E.C.). Anatomical
and mounting evidence in support of the specimen’s identity, genus, sex, and age as presented in the text: C, sternum wooden
replica; D, cranium; E, molar teeth; F, vestigial tusks; G, pelvis. Scale bar in panel A, in white, equals approximately 50 cm.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
cervical vertebrae, and fusion of the spinous processes
of 18th and 19th dorsal vertebrae.
Morphologically, the skeleton is clearly that of an
Asian elephant. Features include the high cranial apex
with a double parietal dome (Fig. 3D), and the charac-
teristic form of the enamel loops of the molars, all in
distinction to Loxodonta (Shoshani, 2000). The jaws
preserve the second molars (M2) in wear and the third
(M3) close to eruption (M5−6 of some authors), indi-
cating (Fig. 3E) an adult animal of c. 25–30 years at
death (Roth & Shoshani, 1988). The specimen’s rela-
tively small size (shoulder height c. 230 cm), vestigial
tusks − tushes − (Fig. 3F), and wide pelvic aperture
(Fig. 3G), indicate that the individual was female
(Lister, 1996).
Analysis of a 741-bp region of mitochondrial
sequence (using DNA derived from a small fragment
of the left humerus) also confirmed the identity of the
Florence specimen as E. maximus. The sequence was
a novel haplotype, closest to the previously reported
haplotype BP (= MDL2), from which it differed at one
nucleotide site (Vidya, Sukumar & Melnick, 2009;
Lei et al., 2012). Although particularly common in Sri
Lanka, haplotype BP is found widely across Asia.
Thus, further resolution of the specimen’s geographi-
cal origin was not possible with current molecular
data, but for nomenclatural stability, the type locality
of E. maximus should continue to be understood to be
the island of Ceylon (‘Zeylonae’ of Linnaeus, 1758).
We established that the Seba foetus was Loxodonta,
and thus that Linnaeus’s original conception of the
taxon E. maximus is composite. Consequently, desig-
nation of a lectotype as the single name-bearing
specimen is necessary to fix the identity of the
species for all future work on its biology, phylogeny,
and conservation.
Exhaustive examination of references cited by
Linnaeus (1758) led us to a detailed description of the
skeleton of an elephant in Florence. We identified
the described skeleton as corresponding to an extant
specimen that proved to be on display at the Natural
History Museum of the University of Florence, and
which both morphological and ancient DNA identified
as an Asian elephant.
Based on this evidence, and in conformity with
Article 74.7 of the ICZN, we hereby designate the
elephant skeleton in Florence, catalogue no. MZUF-
734, as the lectotype of E. maximus Linnaeus, 1758,
to preserve the traditional understanding and appli-
cation of this name to the Asian elephant. Not only
was it amongst the type series but its anatomical
completeness makes it a suitable name-bearing speci-
men from the original type series for this iconic
mammal (Supporting Information S8).
This resolution of nomenclatural inconsistencies
was made possible, 320 years after the first published
description of the hereby designated lectotype speci-
men and 255 years after the naming of the species, by
integrating state-of-the-art experimental research,
based on high-throughput ancient protein and DNA
sequencing, and investigation of the historical litera-
ture, streamlined by digitalization and online avail-
ability of historical texts. The electronic version of
this article, published online ahead of print, is regis-
tered in the ZooBank database ( with the
following LSID:
The authors would like to thank Tim Bouts (Al
Wabra Wildlife Preservation, Qatar), Falko Steinbach
and Akbar Dastjerdi (Animal Health Veterinary
Laboratories Agency, UK), and Stephanie Sanderson
(Chester Zoo) for helping generate the Asian elephant
genomic DNA; Aurélien Ginolhac (Centre for
GeoGenetics) for help in mapping high-throughput
DNA data; Marie-Louise Kampmann and Luise
Ørsted Brandt (Centre for GeoGenetics) for help in
sample preparation; Hannes Schroeder (Centre for
GeoGenetics) for translation from German; Marco
Ferretti (University of Florence) for providing infor-
mation and photographs of the Florence skeleton;
Parc Zoologique de Paris-Vincennes, France, National
Zoological Park, Fort Worth Zoo, Oregon Zoo, St.
Louis Zoo, Ringling Brothers, Florida, for provid-
ing zoo elephant samples; Nicholas J. Georgiadis
(University of Washington) and the governments of
Cameroon, the Central African Republic, Gabon,
Kenya, Republic of the Congo, South Africa, and Tan-
zania for providing modern African elephant samples.
The photos in Figures 1 and S8.1 were taken by
Harry Taylor (NHM, London) and the X-ray by Peter
Mortensen (NRM, Stockholm). Photos in Figure 3
were taken by Saulo Bambi (Florence University).
The original drawing by Stefano Della Bella: ‘Elefante
morto in Firenze nel 1655’, Catalogo Bertini 545,
reported in Figure 3B, is part of the collection of the
Royal Library of Turin, Italy. Its reproduction was
kindly authorized by the ‘Ministero per i Beni e le
Attività Culturali, Direzione Regionale per i Beni
Culturali e Paesaggistici del Piemonte – Biblioteca
Reale di Torino’, prot. n. 0002234. Y. I. and A. R. were
supported by the US Fish and Wildlife Service African
Elephant Conservation Fund Grant AFE-0778-
F12AP01143. This study was funded by the Danish
Independent Research Council ‘Sapere Aude’ and
Danish Basic Research Foundation ‘GeoGenetics’
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
DNRF94 grants and the Swedish Research Council.
A. G. thanks the European Commission for three
grants, originally the High Lat Scheme and now
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Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
S1. Iconography of the sources, other than Seba and Ray, cited by Linnaeus (1758) in the description of Elephas
Figure S1.1. Gesner, C. 1551. Historiae Animalium Lib. 1 de quadrupedibus viviparis. [39], 1104. [11]
pp. Froschover, Zurich.
Figure S1.2. Aldrovandi, U. 1616. De quadrupedibus solidipedibus volumen integrum. 495 pp. Bologna.
Figure S1.3. Jonston, J. 1650. Historiae naturalis de quadrupedibus liber 1. De quadrupedibus Solipedibus.
231 pp., 80 pls. Marianus, Frankfurt.
S2. Albertus Seba, his collection and the fate of his elephant foetus.
S3. Ancient protein sequencing and identification of genus-diagnostic peptides: materials and methods.
Figure S3.1. Tandem spectrum, generated by nanoLC-MS/MS analysis of the ancient foetus sample, supporting
identification of the L. africana version of the genus-diagnostic tryptic peptide E86AALVDVV93NDGVEDLR101.
Figure S3.2. Tandem spectrum, generated from the analysis of the Seba elephant sample, supporting
identification of the L. africana version of the genus-diagnostic tryptic peptide L627YLI630NSPVVR636.
Figure S3.3. Tandem spectrum, generated from the analysis of the Seba elephant sample, supporting identifi-
cation of the L. africana version of the genus-diagnostic tryptic peptide N184M185MFQVLAAEEPTVR198.
Figure S3.4. Tandem spectrum, generated from the analysis of the Seba elephant sample, supporting identifi-
cation of the L. africana version of the genus-diagnostic tryptic peptide V202APLQGV208LPSLLAPLR217.
Table S3.1. Protein recoveries, before and after digestion, for each of the two Seba foetus extracted samples.
Table S3.2. Statistics for MaxQuant MS/MS spectra matching against the Loxodonta africana reference proteome.
Table S3.3. Statistics for MaxQuant MS/MS spectrometry spectra matching against the Elephas maximus
complete protein list.
Table S3.4. Primer sets used to amplify Loxodonta/Elephas DNA regions coding for genus-diagnostic peptides.
Table S3.5. Variable and diagnostic sites across geographically diverse Asian, African forest and African
savannah elephants.
Table S3.6. Primers used to amplify Loxodonta/Elephas diagnostic sites in mitochondrial DNA.
Table S3.7. PCR primers and M13 tails used in this study to attempt amplification of nuclear nucleotide sites
diagnostic between African forest and African savannah elephants (Ishida et al., 2011).
Table S3.8. Evidence supporting the identities of proteins bearing genus-diagnostic tryptic peptides.
Table S3.9. Clade-informative sites overlapping mtDNA sequences generated for the Seba elephant foetus.
S4. John Ray and his tour across part of Europe.
S5. Translation of Ray’s Latin text mentioning the elephant he observed in Florence.
S6. Reconstruction of the fate of the Florence elephant.
Table S6.1. Inventory numbers documenting the presence of the elephant skeleton observed by John Ray in the
Florence Royal Museum since the end of the 18th century.
Table S6.2. Comparison of the measures of the elephant skeleton reported by Targioni Tozzetti (1763) and those
of specimen 734 as measurable today.
S7. Possible identification of the Florence elephant as the performing itinerant elephant known as ‘Hansken’.
Figure S7.1. Rembrandt, An elephant, 1637 (drawing). Vienna, Albertina.
S8. Another extant syntype specimen of Elephas maximus: a partial elephant tooth in Uppsala.
Figure S8.1. Elephas maximus molar fragment (catalogue number UUZM 370) in the Uppsala University
Zoological Museum collection.
© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 222–232
... From this extracted DNA, we attempted to amplify six short DNA fragments (four mitochondrial and two nuclear fragments) by polymerase chain reaction (PCR). The short mitochondrial DNA fragments were selected because they contain diagnostic positions to discriminate between the Asian elephant (genus Elephas) and the African elephants (genus Loxodonta) and may provide some information on the geographic origin of the ivory (Cappellini et al., 2014). In addition, we tried to amplify two nuclear fragments that contain nucleotide sites that are diagnostic for the two African elephant species, which cannot be identified using mitochondrial data. ...
... Ele-CytbF1/Ele-CytbR1 (Cappellini et al., 2014), respectively. One (Bandelt et al., 1999). ...
... additional mitochondrial fragment of 116 bp of the cytochrome b gene was targeted with the primer pair L15123/H15240(Ngatia et al., 2019). One last mitochondrial fragment of 53 bp of the ND5 gene, coding for the NADH dehydrogenase 5 protein, was amplified using the primers Ele-ND5-F3/Ele-ND5-R3(Cappellini et al., 2014).The nuclear fragments, consisting of 4 and 26 bp of the phosphorylase kinase (PHK) and the biglycan (BGN) genes, respectively, were tested using the primer pairs PHK-s1F/PHK-s1R and BGN-s1F2/ BGN-s1R2(Ishida, Demeke, et al., 2011;Ishida, Oleksyk, et al., 2011).Each PCR consisted of a mix of 25 μl with 3 μl of DNA template,1.5 mM of Mg 2+ , 0.2 mM of each dNTP, 0.5 μM of each primer, 0.2 μg/μl of bovine serum albumin, 0.03 units/μl of Platinum Taq DNA Polymerase, and 1 Â PCR buffer (Invitrogen, ThermoFisher). The PCR profiles for the mitochondrial markers consisted of a first step at 94 C for 3 min; a second step of 40 cycles at 94 C for 30 s, primer annealing at 51 C, 52 C, 52 C, and 57 C (for the primer pairs L15123/H15240, Ele-CytbF1/Ele-CytbR1, Ele-ND5-F3 and Ele-ND5-R3, and AFDL3/AFDL4, respectively) for 30 s and 72 C for 15 s;and a final step at 72 C for 7 min. ...
The morphological identification of raw biological material used to produce archaeological artefacts is sometimes difficult or even impossible. In recent years, newly developed biochemical techniques have allowed more reliable identification of exploited animal species, even for otherwise taxonomically undiagnostic fragments, and thus can help pinpoint the geographical origin of the raw material. However, in addition to being costly, these techniques involve destructive sampling. This explains why they are rarely applied to archaeological artefacts, especially those made of precious, imported raw material or those representing intact works of art. Here, we analysed the ancient DNA (aDNA) of a medieval chess piece made of ivory of unknown origin, recovered from a medieval settlement in Jambes (Namur), Belgium. This chess piece was broken during excavation. We took this unfortunate event as an opportunity to perform aDNA extraction, to try to answer three questions: 1) What Proboscidean species does the ivory come from?; 2) Can we establish the geographic origin of the ivory more precisely?; and 3) Does doing so help our understanding of (part of) the trade route followed by the ivory? We sequenced two short fragments of the mitochondrial genome and compared them with publicly available DNA data. This enabled the identification of the raw material as an African elephant (genus Loxodonta). Although the results cannot exclude that the ivory comes from a forest elephant, the recovered DNA sequence is currently found only among savanna elephant DNA records. The ivory likely originates from an eastern or southern African country and was therefore probably transported along the African trade route passing through the Swahili Corridor. However, the precise itinerary followed by this ivory from the African shore of the Mediterranean Sea to Europe, and then to the archaeological site from which it was recovered, remains unknown. Such identification contributes to documenting past trade networks and long-distance exchange.
... This, however, is in tension with the findings of Cappellini et al. (2014). Instead of being a member of the species that we recognize as Elephas maximus today, the elephant foetus clearly belongs to Loxodonta africana. ...
... 638-639). Ray's and Skippon's detailed remarks about the specimen's morphology and anatomy-which included references to its wooden replacement-ribs and toes-enabled Cappellini et al. to match the description to a specimen that is still on display at the Natural History Museum of the University of Florence (for a detailed analysis and translations from Ray's Latin account, see Supplement 4 of Cappellini et al. 2014). ...
... In our opinion, this paper sends a wrong message to taxonomists and to the biological community as a whole. This message is that "problems", that had remained unsolved for centuries because taxonomists were only relying on morphology, can now be solved thanks to modern molecular techniques, and that this will at last salvage taxonomy from its old-fashioned techniques and thinking, to propel it into modernity… In the end, the main purpose of the paper by Cappellini et al. (2014) seems to have been to make the promotion of modern molecular techniques more than to solve a "phantom" nomenclatural problem. (Dubois et al. 2014, p. 58) It is perhaps a bit too brusque to characterize Cappellini et al.'s act of lectotypification as a solution to a "phantom problem," merely because there has largely been agreement among mammalogists about the naming and classification of Asian and African elephants. ...
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What is it to make an error in the identification of a named taxonomic group? In this article we argue that the conditions for being in error about the identity of taxonomic groups through their names have a history, and that the possibility of committing such errors is contingent on the regime of institutions and conventions governing taxonomy and nomenclature at any given point in time. More specifically, we claim that taxonomists today can be in error about the identity of taxonomic groups in a way that Carl Linnaeus (1707–1778), who is routinely cited as the “founder” of modern taxonomy and nomenclature, simply could not be. Starting from a remarkable recent study into Linnaeus’s naming of Elephas maximus that led to the (putative) discovery of a (putative) nomenclatural error by him, we reconsider what it could mean to discover that Linnaeus misidentified a biological taxon in applying his taxon names. Through a further case study in Linnaean botany, we show that his practices of (re)applying names in taxonomic revisions reveal a take on determining “which taxon is which” that is strikingly different from that of contemporary taxonomists. Linnaeus, we argue, adopted a practice-based, hands-on concept of taxa as “nominal spaces” that could continue to represent the same taxon even if all its former members had been reallocated to other taxa.
... Ancient DNA revealed these to belong to three different species, none of which were L. perspicillata (Barbanera et al., 2016). Similarly, Cappellini et al. (2014) used a combination of ancient DNA and proteomics to correct the taxonomic assignment of the original type material of the Asian elephant Elephas maximus. The biomolecular data revealed that this sample, a complete ethanol-preserved fetus originally described by Linnaeus, was in fact an African elephant, Loxodonta sp. ...
... The biomolecular data revealed that this sample, a complete ethanol-preserved fetus originally described by Linnaeus, was in fact an African elephant, Loxodonta sp. (Cappellini et al., 2014). ...
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Museum collections are essential for reconstructing and understanding past biodiversity. Many museum specimens are, however, challenging to identify. Museum samples may be incomplete, have an unusual morphology, or represent juvenile individuals, all of which complicate accurate identification. In some cases, inaccurate identification can lead to false biogeographic reconstructions with cascading impacts on paleontological and paleoecological research. Here we analyze an unusual Equid mandible found in the Far North of the Taymyr peninsula that was identified morphologically as Equus hemionus, an ancestor of present-day Asiatic wild asses. If correct, this identification represents the only finding of a putative Late Pleistocene hemione in the Arctic region, and is therefore critical to understanding wild ass evolution and paleoecology. To confirm the accuracy of this specimen's taxonomic assignment, we used ancient DNA and mitochondrial hybridization capture to identify and place this specimen in the larger equid phylogeny. We find that the specimen is actually a member of E. caballus, the ancestor of domestic horses. Our study demonstrates the utility of ancient DNA to validate morphological identification, in particular of incomplete, otherwise problematic, or taxonomically unusual museum specimens.
... Perhaps, the most striking example among vertebrates is that of the type material of the Asian elephant, Elephas maximus Linnaeus, 1758, which was found to contain a specimen of the African elephant, Loxodonta sp. based on a morphological re-examination and analyses of ancient DNA (Cappellini et al. 2014). Furthermore, several Linnean fish species were found to be composite taxa based upon a thorough reappraisal of the type series (Fernholm and Wheeler 1983). ...
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India represents a tropical biodiversity hotspot, harbouring a unique and largely endemic fauna of freshwater mussels (Bivalvia: Unionidae). Unfortunately, the type series of many freshwater mussel taxa historically described from this country are thought to be lost. Here, we report on the rediscovery of the type series of Parreysia corrugata (O.F Müller, 1774) (Parreysiinae: Parreysiini) in the Natural History Museum of Denmark (NHMD), Copenhagen. It contains four syntypes (complete shells), which were examined by means of morphological and geometric morphometric techniques. We show that the nominal taxon Parreysia corrugata was established based on a composite type series, because one of the syntypes belongs to another species, Potomida semirugata (Lamarck, 1819) (Gonideinae: Lamprotulini), originating from the Middle East. To fix the taxonomic identity of the Indian species, a syntype (lot NHMD 916309) that was most likely illustrated by O.F. Müller in 1779 is designated here as the lectotype of Parreysia corrugata. The lectotype, paralectotypes and corresponding labels are illustrated in detail. Our new findings clarify the taxonomic concept of Parreysia corrugata and contribute to the further development of the Indian Unionidae taxonomy.
... The extracted proteins are digested into peptides using an enzyme, commonly trypsin. Several digestion methods have been reported, such as liquid digestion (Horn et al., 2019;Sawafuji et al., 2017), filter assisted sample preparation (FASP) (Cappellini et al., 2014;Kostyukevich et al., 2018), solid digestion of demineralized bones (Cleland, 2018b), and microwave digestion (Colleary et al., 2020). Recently a single-pot, solid-phase-enhanced sample preparation (SP3), an effective method allowing for collagen and non-collagenous proteins (NCPs) analyses together with the removal of co-extracted humic compounds, has been proposed (Cleland, 2018a). ...
Ancient preserved molecules offer the opportunity of gaining a deeper knowledge on their biological past. However, the development of a proteomic workflow remains a challenge. The analysis of fossils must involve a low quantity of material to avoid damaging the samples. In this study an enhanced proteomic protocol was applied to 5‐milligram samples of about 130,000‐year‐old mammalian bones ranging from the end of the Middle Pleistocene up to the earlier Upper Pleistocene, excavated from Scladina Cave (Sclayn, Belgium). Using sequence homology with modern sequences, a biological classification was successfully achieved and the associated taxonomic ranks to each bone were identified consistently with the information gained from osteomorphological studies and palaeoenvironmental and palaeodietary data. Amino acid substitutions on collagens were identified, thus providing new information on extinct species sequences and helping in taxonomy‐based clustering. Considering samples with no osteomorphological information, such as two fragments of bone retouchers, proteomics successfully identified the families providing paleontologists new information on these objects. Combining osteomorphology studies and amino acid variations identified by proteomics, one of the retouchers was potentially identified as belonging to the Ursus spelaeus species.
... While the example is contrived, in practice such type reinterpretations are not infrequent (e.g., Ribot et al. 1996;Woodley et al. 2011;Cappellini et al. 2013;Laloy et al. 2013;Witteveen 2015). Reassessments of the taxonomic identity of type specimens are very frequently involved in determining heterotypic synonymy. ...
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We utilize an Answer Set Programming (ASP) approach to show that the principles of nomenclature are tractable in computational logic. To this end we design a hypothetical, 20 nomenclatural taxon use case with starting conditions that embody several overarching principles of the International Code of Zoological Nomenclature; including Binomial Nomenclature, Priority, Coordination, Homonymy, Typification, and the structural requirement of Gender Agreement. The use case ending conditions are triggered by the reinterpretation of the diagnostic features of one of 12 type specimens anchoring the corresponding species-level names. Permutations of this child-to-parent reassignment action lead to 36 alternative scenarios, where each scenario requires 1-14 logically contingent nomenclatural emendations. We show that an ASP transition system approach can correctly infer the Code-mandated changes for each scenario, and visually output the ending conditions. The results provide a foundation for further developing logic-based nomenclatural change optimization and compliance verification services, which could be applied in globally coordinated nomenclatural registries. More generally, logic explorations of nomenclatural and taxonomic change scenarios provide a novel means of assessing design biases inherent in the principles of nomenclature, and thus may inform the design of future, big data-compatible identifier systems for systematic products that recognize and mitigate these constraints.
... Identification of collagen peptides has successfully been reported from very old and more recent bone material; see, for instance: Asara (2007) and Buckley et al. (2010). Cappellini et al. (2013) demonstrated the value of a combined genomics and proteomics approach by elucidating the correct identity of a nearly 300-yearold ethanol-preserved elephant foetus. The same group was able to retrieve peptides from 126 unique proteins extracted from a 43 290-year-old femur of a woolly mammoth [Mammuthus primigenius (Blumenbach, 1977); Cappellini et al., 2012] and very recently used proteome sequences from 1.77 million years old enamel to investigate the phylogenetic relationships of the Eurasian Pleistocene Rhinocerotidae (Cappellini et al., 2018). ...
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We used proteomic profiling to taxonomically classify extinct, alongside extant bird species using mass spectrometry on ancient bone-derived collagen chains COL1A1 and COL1A2. Proteins of Holocene and Late Pleistocene-aged bones from dodo (Raphus cucullatus) and great auk (Pinguinus impennis), as well as bones from chicken (Gallus gallus), rock dove (Columba livia), zebra finch (Taeniopygia guttata) and peregrine falcon (Falco peregrinus), of various ages ranging from the present to 1455 years old were analysed. HCl and guandine-HCL-based protein extractions from fresh bone materials yielded up to 60% coverage of collagens COL1A1 and COL1A2, and extractions from ancient materials yielded up to 46% coverage of collagens COL1A1 and COL1A2. Data were retrieved from multiple peptide sequences obtained from different specimens and multiple extractions. Upon alignment, and in line with the latest evolutionary insights, protein data obtained from great auk grouped with data from a recently sequenced razorbill (Alca torda) genome. Similarly, protein data obtained from bones of dodo and modern rock dove grouped in a single clade. Lastly, protein data obtained from chicken bones, both from ancient and fresh materials, grouped as a separate, basal clade. Our proteomic analyses enabled taxonomic classification of all ancient bones, thereby complementing phylogenetics based on DNA. ADDITIONAL KEYWORDS: ancient proteins-bird taxonomy-dodo-extant birds-great auk-palaeoproteomics-phylogeny.
... Extraction protocol A (filter-aided sample preparation). Tryptic peptides were generated using a filter-aided sample preparation approach 38 , as previously performed on ancient samples 39 . Extraction protocol B (GuHCl solution and digestion). ...
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The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa¹. However, the irreversible post-mortem degradation² of ancient DNA has so far limited its recovery—outside permafrost areas—to specimens that are not older than approximately 0.5 million years (Myr)³. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I⁴, and suggested the presence of protein residues in fossils of the Cretaceous period⁵—although with limited phylogenetic use⁶. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7–9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)¹⁰. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck’s rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel—which is the hardest tissue in vertebrates¹¹, and is highly abundant in the fossil record—can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.
The latest developments in archaeology and palaeontology have seen the ‘explosion’ of ‐omics tools. Protein biogeochemistry has morphed from an amino acid‐based analytical approach to a ‘palaeoproteomic’ one, opening up exciting possibilities for retrieving phylogenetic information from fossil biominerals. This sudden expansion, however, can also mean that the broad knowledge accumulated in the past 70 years of ‘traditional’ studies may not always be taken into account. This chapter gives a brief overview of mass spectrometry‐based ancient protein research, including novel applications to invertebrate systems (mollusc shells), and concludes with a reflection on the complex and fruitful legacy of palaeobiogeochemistry
There are four main types of biominerals that are, at present, especially relevant for ancient protein studies with applications to archaeology, palaeontology and earth sciences: these are bone (and other collagenous hard tissues), teeth, mollusc shells and bird eggshell. Additionally, coral, foraminifera, brachiopods and arthropods can also frequently be found in the fossil record and have the potential to be targeted in future studies on ‘fossil’ biomineralized proteomes. This chapter briefly reviews the main characteristics of each of these materials and of their proteomes, as well as their occurrence in archaeological and palaeoenvironmental sites.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1, 2, 3, 4.
We describe a simple method for extracting polymerase chain reaction‐amplifiable DNA from ancient bones without the use of organic solvents. Bone powders are digested with proteinase K, and the DNA is purified directly using silica‐based spin columns (QIAquick™, QIAGEN). The efficiency of this protocol is demonstrated using human bone samples ranging in age from 15 to 5,000 years old. Am J Phys Anthropol 105:539–543, 1998. © 1998 Wiley‐Liss, Inc.
This biography of John Ray, the seventeenth-century naturalist, was first published in 1942 at the height of the Second World War. It was written by Charles Raven, an eminent theologian who shared Ray's deep respect for intellectual integrity, honest exploration of the natural world, and the value of both theology and scientific endeavour. More than a superb history, this offers an opportunity to reassess the pivotal contributions of a brilliant but often undervalued scientist. Ray's major publications were written in Latin; Raven's linguistic skills – coupled with his passion for natural history – made him ideally suited to interpret Ray's scientific legacy. Raven reviews Ray's academic and scientific careers in the context of the dramatic social upheavals of his time. He evaluates the remarkable long-term and widespread influence of Ray's work on the development of science, alongside the significance of his final book, The Wisdom of God.
THE last work that Bernini and his assistants undertook for Pope Alexander VII is the monument erected in 1667 on the Piazza della Minerva in Rome (Fig. 1). It displays a small Egyptian obelisk that Bernini placed upon the back of an almost life-size marble elephant, who in turn stands on a high and narrow pedestal. The obelisk is crowned by the insignia of the Pope (Chigi mountains and Chigi star) with the Cross of Christ on top. Two plaques on the plinth bear dedicatory inscriptions. The monument occupies the approximate center of the Piazza, opposite the main entrance of the west façade of S. Maria sopra Minerva, with the axis of the elephant's body facing south.